A Workflow to Increase Verification Rate of Chromosomal Structural Rearrangements Using High-Throughput Next-Generation Sequencing

Abstract

Somatic rearrangements, which are commonly found in human cancer genomes, contribute to the progression and maintenance of cancers. Conventionally, the verification of somatic rearrangements comprises many manual steps and Sanger sequencing. This is labor intensive when verifying a large number of rearrangements in a large cohort. To increase the verification throughput, we devised a high-throughput workflow that utilizes benchtop next-generation sequencing and in-house bioinformatics tools to link the laboratory processes. In the proposed workflow, primers are automatically designed. PCR and an optional gel electrophoresis step to confirm the somatic nature of the rearrangements are performed. PCR products of somatic events are pooled for Ion Torrent PGM and/or Illumina MiSeq sequencing, the resulting sequence reads are assembled into consensus contigs by a consensus assembler, and an automated BLAT is used to resolve the breakpoints to base level. We compared sequences and breakpoints of verified somatic rearrangements between the conventional and high-throughput workflow. The results showed that next-generation sequencing methods are comparable to conventional Sanger sequencing. The identified breakpoints obtained from next-generation sequencing methods were highly accurate and reproducible. Furthermore, the proposed workflow allows hundreds of events to be processed in a shorter time frame compared with the conventional workflow.

Structural variation in cancer genomes & the identification of personalized DNA-based cancer biomarkers

“…Furthermore, this workflow is applicable to both long mate pair and pair end sequencing data as the qSV tool has the ability to identity structural rearrangements from SOLiD and Illumina sequencing platforms. This work has been published in BioTechniques Report as a method article in early 2014 (Quek et al, 2014).…”

“…BioTechniques 57:3-38. A workflow to increase verification rate of chromosomal structural rearrangements using high throughput next generation sequencing (Quek et al, 2014).…”

“…An additional 64 somatic rearrangements breakpoints from three tumours were verified using amplicon deep sequencing as previously described in Chapter 2 (Quek et al, 2014). In brief, the verification workflow of breakpoints combines the pooling strategy of amplicons with benchtop sequencing and standard bioinformatics techniques such as batch primer design, de novo assembly of sequencing data, and automated BLAT to confirm the breakpoints of the somatic rearrangements.…”

“…QCMG is part of the ICGC (International Cancer Genome Consortium) and in 2014, we completed the sequencing and analysis of 392 primary pancreatic ductal adenocarcinomas, 100 pancreatic neuroendocrine tumours and 93 ovarian tumours. During this process, the laboratory not only generated and analysed a large volume of sequencing data but also developed several bioinformatics tools to facilitate the genomics analysis of these tumours (Kassahn et al, 2013;Quek et al, 2014;Song et al, 2012). The sequence data was analysed by the QCMG to determine somatic base pair substitutions, indels, copy number changes and chromosomal structural rearrangements.…”